title: The CD47-binding peptide of thrombospondin-1 induces defenestration of liver sinusoidal endothelial cells.
abstract: OBJECTIVE
  A fenestrated phenotype is characteristic of liver sinusoidal endothelial cells (LSECs), but liver sinusoids become defenestrated during fibrosis and other liver diseases. Thrombospondin-1 (TSP1) is a matrix glycoprotein with pro-fibrotic effects, and the CD47-binding fragment of TSP1 also has anti-angiogenic effects in endothelial cells. We hypothesized that the CD47-binding fragment of TSP1 could induce defenestration in LSECs through the Rho-Rho kinase (ROCK)-myosin pathway.
  
  
  METHODS
  Freshly isolated rat LSECs were treated with TSP1 or CD47-binding peptides of TSP1. LSEC fenestration was assessed with scanning electron microscopy, and myosin phosphorylation was assessed with immuno-fluorescence.
  
  
  RESULTS
  Treating LSECs with TSP1 caused a dose-dependent loss of fenestrae, and this effect could not be blocked by SB-431542, the TGF-β1 receptor inhibitor. A CD47-binding fragment of TSP1, p4N1, was able to induce defenestration, and a CD47-blocking antibody, B6H12, was able to suppress p4N1-induced defenestration. The p4N1 fragment also caused contraction of fenestra size, correlated with an increase in myosin activation. Pretreatment with Y-237642 (a ROCK inhibitor) prevented p4N1-induced myosin activation and fenestrae decrease. Simvastatin has also been shown to antagonize Rho-ROCK signalling, and we found that simvastatin pretreatment protected LSECs from p4N1-induced myosin activation and defenestration.
  
  
  CONCLUSIONS
  We conclude that CD47 signals through the Rho-ROCK-myosin pathway to induce defenestration in LSECs. In addition, our results show that simvastatin and Y-237642 have a beneficial impact on fenestration in vitro, providing an additional explanation for the efficacy of these compounds for regression of liver fibrosis.
authors.affiliation: Mechanobiology Institute, National University of Singapore, Singapore.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Venkatraman
authors.forename: Lisa
authors.initials: L
authors.lastname: Tucker-Kellogg
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D051928:CD47 Antigen / Q000235:genetics / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D007091:Image Processing, Computer-Assisted; D008099:Liver / Q000166:cytology*; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D009218:Myosins / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D051928:CD47 Antigen; D054460:rho-Associated Kinases; D009218:Myosins
keywords: capillarization; discontinuous endothelium; mechanical signal transduction; myosin light chain